The present invention relates to luminescence assays based on transfer of excitation energy from a donor species to an acceptor species.
The present invention relates to luminescence assays based on transfer of excitation energy from a donor species to an acceptor species.
Fluorescence, phosphorescence and related technologies (herein referred to as “luminescence” to include in this context all processes where energy is emitted subsequent to an excitation process triggered by absorption of electromagnetic radiation) are now widely used in a variety of analytical schemes. Luminescent materials are used as tracers on the basis of the high detection sensitivity that can be achieved, but are also used as environmentally responsive “probes” to monitor local conditions, such as pH, ion concentrations, oxygen tension etc. Luminescent species can also be used to detect and sometimes quantify the proximity of an agent which is able to modify the emission process on close approach or contact.
It is well known that energy can be transferred by a variety of means from an excited species (the “donor”) to a second species able to act as an energy acceptor. One of the most common examples of such transfer involves a radiationless process known as resonance energy transfer, the efficiency of which usually has an inverse sixth power dependence on distance between donor and acceptor. Distance-dependent energy transfer between donor and acceptor species has been used in a variety of analytical and assay formats. An analyte might be detected on the basis of its ability to bind to a site where it can function as one member of the energy transfer donor-acceptor pair. Alternatively, the assay might be conducted in a competitive format where the analyte displaces a labelled analogue from a site and the displacement can be detected and quantified by changes in energy transfer between the site and the analogue. One common type of assay involves the detection of an analyte on the basis of its ability to bind two recognition molecules such as antibodies simultaneously. In this “sandwich” format, the proximity of the two bound species can be determined by energy transfer between labels bound to the antibodies. A recent example of an energy transfer assay using a luminescent cryptate of long emissive lifetime to transfer energy to a short-lived acceptor species is given by G. Mathis (“Rare Earth Cryptates and Homogeneous Fluoroimmunoassays with Human Sera”, Clinical Chemistry, Volume 39(9), 1953-1959, (1993)).
Another approach to the assay of enzymes or similar catalytic species (e.g. ribozymes) relies on the ability of the analyte to cleave a chemical bond linking an energy transfer donor to an acceptor species. For example, a protease can be assayed by monitoring the decrease in energy transfer efficiency between donor and acceptor linked together by a peptide fragment. As the linkage is broken the donor and acceptor become separated and efficient transfer of energy is no longer possible. Conversely, an analyte able to initiate chemical bond formation might be assayed on the basis of increase in energy transfer between a suitable donor and acceptor pair as they become linked together. These and similar methods of assay based on transfer of excitation energy are well known in the literature.
For a general discussion of analytical applications of luminescence in Biology see for example “Applications of Luminescence Spectroscopy to Quantitative Analyses in Clinical and Biological Samples”, P. Froehlich in “Modem Fluorescence Spectroscopy” Volume 2, E. L. Wehry ed., Plenum, N.Y. (1976). Details of a variety of fluorescence assay procedures are given in “Principles and Practice of Fluoroimmunoassay Procedures”, D. S. Smith, M. Hassan and R. D. Nargessi in Volume 3 of the same series.
Most assays involving energy transfer processes are relatively insensitive and can have limited dynamic range. One problem with all luminescence detection is the sensitivity limit set by background. In this context, background might be unwanted luminescence from sample or container, elastically or inelastically scattered exciting light which is not totally rejected by optical filters, luminescence from filters themselves or from other optical elements or any other source of electromagnetic radiation detectable by the measuring apparatus. In absence of background, luminescence methods are commonly able to detect single atoms or molecules. Most analytical luminescence measurements fall short of this sensitivity by orders of magnitude and therefore methods to minimise background are very important in practical applications. Unfortunately, luminescence energy transfer is commonly troubled by background from a variety of causes.
In a practical measurement of energy transfer, ideally the donor is excited by radiation not absorbed by the acceptor. The transfer is detected either by measurement of quenching of donor emission (or related parameters such as change in donor decay rate), or by the sensitised emission from the acceptor if this is also luminescent. Ideal conditions are rarely achieved however and the following potential difficulties and sources of background must be considered:                It is often difficult to avoid some level of direct excitation of the acceptor species by radiation used to excite the donor. In assays where the acceptor is luminescent, this means that there is usually a background of luminescence from the acceptor, even in the absence of any energy transfer from the donor and this in turn limits dynamic range of any assay using the donor-acceptor pair.        Exciting radiation is capable of exciting luminescence from impurities or other unwanted components of the sample.        The donor might emit some radiation at a wavelength which overlaps with the passband of the optical filters used to isolate emission from the acceptor, so that a signal is detected in absence of energy transfer.        
These and related problems limit the scope, sensitivity and dynamic range of assays based on energy transfer processes.